With the intensive use of antimicrobial agents, drug resistance of pathogens to antimicrobial agents is increasing, and the therapy for infection due to resistant bacteria becomes a global problem. The pathogens that cause the clinical bacterial infection include aerobes and anaerobe, and are classified as gram-positive and gram-negative bacteria. Each class includes coccus and rod. Aerobic gram-positive coccus and gram-negative rod are two kinds of the most frequently encountered pathogens. Due to the difference of cell wall and other structures between gram-positive and gram-negative bacteria, the mechanism of drug action and drug resistance displays the significant difference, and the clinical therapy to the two kinds of bacteria are also very different.
Antibiotics are the most frequently used antimicrobial agents. They play an important role in controlling, preventing and treating the infectious diseases. However, since antibiotics were introduced in 1939, especially in recent 10 years, the drug resistance of bacteria is increasing rapidly, and the nosocomial infection is more and more serious.
The emergence of all kinds of different resistant bacteria can cause a serious problem. For example, it can result in the therapy failure, the increase of the complicating disease, the infection recrudescence, the delay of the hospital time, and the increase use of expensive antibiotics and other drugs. The global spread of resistant bacteria is increasing with the development of international trade. Studying of the emergence of resistant bacteria and its occurrence mechanism, and understanding and using the correct detection methods are the key points to in time detect the resistant strains, and to efficiently prevent and control the spread of the resistant strains. Timely and accurate detection of resistant bacteria is important to the rational use of antimicrobials and the development of new antimicrobial agents.
The resistance mechanism of gram-negative bacteria is very complex, including four kinds of major resistance mechanisms. The multi-drug resistance also occurs due to the synergetic effect of different resistance mechanisms.
The Modification or Hydrolysis of Enzymes to Antibiotics
β-lactamase, inactivating the antibiotics by hydrolyzing β-lactam ring, is the most important mechanism for most bacteria to resist β-lactamase, and is the resistance mechanism of about 80% resistant bacteria. More than 200 kinds of β-lactamases have been found. β-lactamase can hydrolyze the β-lactam four-member ring of penicillins, cephalosporins, carbapenems (imipenem and meropenem) and monobactams (aztreonam).
Outer Membrane Impermeability
The membrane porin of gram-negative bacteria is narrow, and can form the effective natural barrier to the macromolecules and hydrophobic compounds. The loss of membrane porin is a major cause of the decrease of membrane impermeability that causes bacterial drug resistance, for example, the loss of OprD2 membrane porin results in the resistance against imipenem in Pseudomonas aeruginosa (Huang H, Jeanteur D, Pattus F, Hancock R E. 1995. Membrane topology and site-specific mutagenesis of Pseudomonas aeruginosa porin OprD. Mol. Microbiol. 16(5):931-41.) and Klebsiella pneumoniae (Martinez-Martinez L, Pascual A, Hernandez-Alles S, Alvarez-Diaz D, Suarez A I, Tran J, Benedi V J, Jacoby G A. 1999. Roles of beta-lactamases and porins in activities of carbapenems and cephalosporins against Klebsiella pneumoniae. Antimicrob Agents Chemother. 43(7):1669-73).
Active Efflux Pumps
Active efflux is now known to play a major role in the resistance of many species to antibacterial agents, effluxing many different antibiotics by the synergism of several membrane proteins, and resulting in bacterial multi-drug resistance. Four active efflux pumps are detected in Pseudomonas aeruginosa responsible for multiple drug resistance [Masuda N, Sakagawa E, Ohya S. 1995. Outer membrane proteins responsible for multiple drug resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 39(3):645-9.]. Among them, only mexAB-oprM is constitutively expressed, and is the basis of multidrug resistance in P. aeruginosa. 
Alteration of the Antibiotic Target
β-lactam antibiotics bind specifically with PBPs on the cell membrane, and kill bacteria by inhibiting the synthesis of peptidoglycan. The antibiotics loose their functions due to the alteration of PBPs or production of new functional PBPs, resulting in the drug resistance. Since the β-lactamases and outer membrane impermeability play an important role in gram-negative bacteria, the function of PBPs in drug resistance is not significant in gram-negative bacteria.
β-Lactamases
β-lactamases (E.C.3.5.2.6) are the enzymes that can hydrolyze specifically β-lactam ring, and are the leading cause of resistance to β-lactam antibiotics among gram-negative bacteria, accounting 80% in all the resistance mechanisms. β-lactamses are a protein family consisting of many kinds of enzymes. The genes of these proteins exist on the bacterial chromosome or plasmids. The penicillinase that can hydrolyze penicillin was reported followed the use of penicillin nearly at the same time [Abraham E P, Chain E. 1940. An enzyme from bacteria able to destroy penicillin. Nature. 146: 837]. Currently the remarkable problem in gram-negative bacteria is the resistance to the third generation cephalosporin and the new extended-spectrum beta-lactam antibiotics. The major mechanism is the production of extended-spectrum beta lactamases (ESBLs) and Bush group of β-lactamases in bacteria. The active site of penicillinase and AmpC lactamase contains a serine, which attack the carboxyl group of β-lactam as the nucleophilic agent, forms the acyl-enzyme intermediate, then β-lactam ring is destroyed and inactive by the action of water molecules. The active site of mental β-lactamase is bivant Zn atom.
So far more than 200 kinds of different β-lactamases have been reported. They are different in the origin, hydrolytic spectrum, susceptibility to inhibitors and the structures, thus resulting in the difficulty for the systermatic classification. Seven classification schemes have been proposed. Among them, the functional classification scheme of β-lactamases proposed by Bush, Jacoby and Medeiros [Bush, K., G. A. Jacoby, and A. A. Medeiros. 1995. A functional classification scheme for β-lactamases and its correlation with molecular structure. Antimicrob. Agents Chemother. 39:1211-1233.] is more practical and perfect, which defines the following four groups according to their substrate and inhibitor profiles.
Group 1 cephalosporinases belong to class C enzyme and are not well inhibited by clavulanic acid. These enzymes can hydrolyze preferably cephalosporin and hydrolyze penicillin weakly. Group 1 enzymes are encoded by ampC genes located on the chromosome, and are called AmpC beta-lactamase. The characteristic of the recently found plasmid-mediated AmpC is same as the chromosomal AmpC enzymes, therefore are classified as group 1 enzymes.
Group 2 penicillinases, cephalosporinases, and broad-spectrum β-lactamases belong to class A or D enzymes that hydrolyze mostly penicillin and are generally inhibited by active site-directed β-lactamase inhibitors (except 2br subclass). In addition, extended-spectrum beta lactamases (ESBLs) belong to 2be subclass and can hydrolyze the third generation of cephalosporin.
Group 3 metallo-β-lactamases, also called carbapenemases, belong to class B, including IMP- and VIM-type β-lactamases. Metallo-β-lactamases are located on the chromosome or the plasmids. Their active site contains two Zn atoms, and can hydrolyze almost all β-lactams, and they are poorly inhibited by inhibitor clavulanic acid. However they can be inhibited by EDTA and p-chloromercuribenzoate (pCMB).
Group 4 penicillinases can hydrolyze penicillin and are not well inhibited by clavulanic acid.
The most important β-lactamases in the clinic are extended-spectrum beta lactamases (ESBLs) and cephalosporinase (AmpC).
The Methods for Detection of ESBLs and AmpC
Disc diffusion method: At least two kinds of discs containing cefpodoxime, ceftazidime, aztreonam, cefotaxime or ceftriaxome are used in the initial screening test. Mueller-Hinton agar with discs is inoculated. After incubation, the inhibition zone diameter is measured and the susceptibility is estimated according to Clinical and Laboratory Standards Institute (CLSI) guideline. The CLSI phenotypic confirmatory method for ESBL production uses the discs containing 30 μg of cefotaxime and ceftazidime alone and in combination with 10 μg of clavulanate. Mueller-Hinton agar is inoculated, and discs containing the standard 30 μg of ceftazidime, or cefotaxime are placed 15 mm from the ceftazidime or cefotaxime disc with 10 μg of clavulanic acid. After incubation, an enhanced zone of inhibition (≧5 mm) between any one of the β-lactam discs and the clavulanic acid disc is interpreted as presumptive evidence for the presence of an ESBL. The sensitivity of the method is 84.4%.
Double disc synergy test: A disc containing enzyme inhibitor (such as amoxicillin-clavulanate) is placed in proximity to a disc containing the third generation of cephalosporin (such as ceftazidime, cefotaxime or ceftriaxome) or aztreonam. The clavulanate in the amoxicillin-clavulanate disc diffuses through the agar and inhibits the β-lactamase surrounding the ceftazidime disc. Enhancement of the zone of the ceftazidime disc on the side facing the amoxicillin-clavulanate disc is interpreted as the presence of an ESBL. The sensitivity of the method is about 79%.
Determination of minimum inhibition concentration (MIC): At least two kinds of antibiotics including cefpodoxime, ceftazidime, aztreonam, cefotaxime or ceftriaxome are used in the initial screening test. MH broth is diluted (standard broth dilution method), and the isolate with the MIC of ceftazidime, cefotaxime or ceftriaxome or aztreonam more than 2 mg/ml is considered as the ESBL-producing isolate. The confirmatory test is performed according to the CLSI guideline. MH broths with cefotaxime (or ceftazidime) alone and with cefotaxime (or ceftazidime) plus clavulanate (with a fixed concentration of 4 mg/L) are diluted respectively. The difference of the MIC of cefotaxime (or ceftazidime) alone and with clavulanate more than 8 folds is interpreted as presumptive evidence for the presence of an ESBL.
Etest: Etest ESBLs strip contains ceftazidime on one end, ceftazidime plus clavulanate on another end, and the antibiotic concentrations on the ends are highest, decreasing successively toward the middle. MH agar with E-test strip is inoculated and cultured for 18-24 hours, then measure the MIC on the inhibition lines. The difference of the MIC of ceftazidime alone and with clavulanate more than 4 folds is interpreted as presumptive evidence for the presence of an ESBL. This method is easy-operated and simple, but the cost of the strips is very expensive.
Three dimensional test: Lawn cultures of the susceptible isolate such as E. coli ATCC 25922 are prepared on Mueller-Hinton agar plates and cefoxitin 30 μg discs are placed on the plate. Linear slits are cut using a sterile surgical blade 3 mm away from the cefoxitin disc and the isolate culture or the extract is loaded. The plates are kept upright for 5-10 min until the solution dried, and are then incubated at 37° C. overnight. The disturbance of the inhibition zone of the susceptible strain by the tested culture or the extract is interpreted as positive. The sensitivity of this method is 95%. However, the method is labor-intensive, and the expert is needed for operation.
The above phenotypic detection method (except three dimensional test) only can be used to detect E. coli, K. pneumoniae and K. oxytoca, and can not be used for other Enterobacteriaceae, also can not be used to detect AmpC enzymes. Three dimensional test can detect AmpC enzymes, but it is difficult to perform in the clinical laboratory.
Besides the said phenotypic resistance detection methods, some molecular methods can also be used to detect ESBLs, such as detection of pI of ESBLs (isoelectric focusing, IEF), analysis of the hydrolytic spectrum of enzyme for typing or PCR amplification. The advantages of the molecular methods are rapidness and accuracy. When the MIC is the critical value, molecular methods can provide useful information for clinical therapy. However, the detection target of the traditional molecular method is few, thus the practical application is not good.
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